Mononuclear phagocytes (MO) produce urokinase-type plasminogen activator (uPA) and also express a specific cell-surface receptor for urokinase, uPAR. The concomitant expression of these proteins provides a mechanism by which MO can degrade extracellular matrix proteins during directed cell migration. In this study, we sought to determine if uPAR plays a role in MO chemotaxis that is distinct from its role in matrix proteolysis. Exposing adherent monocytes to a chemotactic gradient causes plasma membrane uPAR to localize strongly to the leading edge of cell migration. Adherence alone or exposure to FMLP had no effect on uPAR expression. Using Boyden chamber chemotaxis assays, we demonstrate that treating mononuclear cells with an anti-uPAR mAb (either as an intact mAb or Flab'12) ablates chemotaxis induced by FMLP and monocyte chemotactic peptide-1 (P < 0.001). Inactivating the catalytic activity of uPAR-bound uPA had no effect on chemotaxis. Similarly, blocking uPAR expression with an antisense oligonucleotide to uPAR completely ablates chemotaxis, but blocking uPA expression with an antisense oligonucleotide to uPA has a minimal effect. We therefore demonstrate that expression and unimpeded function of uPAR plays an obligate role in M6 chemotaxis by mechanisms that are largely independent of its ligand, uPA. Combined with its known role in mediating pericellular proteolysis, these observations demonstrate that uPAR is essential for both locomotion and traversing tissue barriers during MO migration. (J. Clin. Invest. 1994.

Urokinase receptors (uPAR; CD87) form complexes with complement receptor 3 (CR3) (CD11b/CD18), a ␤ 2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogenadherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a ␤ 1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43 Ϯ 9% ( P Ͻ 0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% ( P Ͻ 0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.

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3040 Poster Board II-1016 The physiologic function of factor XII (XII) is not known. New interest in XII has occurred because XII KO mice are protected from thrombosis. Exposed arterial collagen, aggregated protein, RNA, and platelet polysomes are recently recognized entities in developing thrombus that promote XII autoactivation to XIIa increasing thrombus formation, independent of a role in hemostasis. However, these observations do not indicate a constitutive, physiologic function for XII. We sought a physiologic function for zymogen XII. XII has been recognized to stimulate MAP kinase. Cleaved high molecular weight kininogen (HKa) is known to be antiangiogenic. Further, XII and HK mutually block each others binding to endothelial cells. Both XII and HKa bind urokinase plasminogen activator receptor (uPAR) at an overlapping site. We investigated if XII stimulates cells by interacting with uPAR and if this activity influences angiogenesis. XII (3-200 nM) with 0.05 mM Zn ion induces ERK1/2 (MAPK44 and 42) and Akt (Ser473) phosphorylation in endothelial cells. XII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. The uPAR signaling region for XII is on domain 2 adjacent to uPAR's integrin binding site. HKa or peptides from HKa's domain 5 inhibit XII-induced ERK1/2 and Akt phosphorylation. A beta-1-integrin peptide that binds uPAR, antibody 6S6 to beta-1-integrin, or the EGFR inhibitor AG1478 blocks XII-induced phosphorylation of ERK1/2 and Akt. XII induces endothelial cell proliferation and 5-bromo-2'deoxy-uridine incorporation. XII stimulates aortic sprouting in normal but not uPAR deficient mouse aorta and this mechanism is blocked by PD98059, LY294002, AG1478, or HKa. XII also induces angiogenesis in matrigel plugs. Finally, XII knockout mice have reduced constitutive and wound-induced blood vessel number on initial biopsy and 9 days after wound healing, respectively. In sum, XII initiates outside-in signaling mediated by uPAR, beta-1-integrin, and the EGFR leading to HUVEC proliferation, growth, and angiogenesis. XII is a constitutive proangiogenic protein. Disclosures No relevant conflicts of interest to declare.

During recruitment, leukocytes respond to chemotaxins and traverse matrix barriers. Urokinase-type plasminogen activator (uPA), bound to its receptor (uPAR; CD87) facilitates plasmin formation, which promotes matrix proteolysis. Polymorphonuclear leukocytes (PMNs) are critical to the inflammatory response and express both uPA and CD87. To determine whether uPA and CD87 are required for PMN chemotaxis, PMNs were pretreated with an anti-CD87 monoclonal antibody (mAb), a neutralizing anti-uPA mAb, or uPA. PMN chemotaxis was profoundly suppressed by the anti-CD87 mAb but was unaffected by anti-uPA mAb or uPA. The role CD87 plays in chemotaxis may be related to its ability to associate with CR3. CD87/CR3 coupling can be disrupted by specific saccharides. The same saccharides that disrupt CD87/CR3 coupling (NADG, D-mannose, and mannoside) inhibit PMN chemotaxis. We conclude that CD87 plays a crucial role in PMN chemotaxis in vitro that is independent of uPA enzyme activity but may be related to the ability of CD87 to interact with CR3.